Nonisotopic immunoassays are widely used in both clinical and research contexts for the determination of both the presence and quantity of analytes such as proteins, nucleotide sequences, drugs, steroids, etc. Nonisotopic immunoassays can be divided into two types: heterogeneous assays and homogeneous assays.
In heterogeneous assays, a solid support (e.g., beads, a column) is used; some of the labeled reagent becomes bound to the support, while the remainder does not. A procedure is required to separate bound and free labeled reagent.
In homogeneous assays, no separation is required, thus eliminating the need for an additional step. There are at least five major types of homogeneous immunoassays routinely used. Three of these fall into a subcategory of fluorescence immunoassays: substrate-labeled fluorescence immunoassay (SLFIA); Forster energy transfer (FITC) and fluorescence polarization immunoassay (FPIA).
Fluorescence polarization immunoassays can be used to measure small quantities of substances (e.g., in the nanogram-per-milliliter range). They make use of the fact that molecules can exist in a ground or lowest energy state and, after exposure to incident radiation, an excited or higher energy state. Absorption of energy from this source results in promotion of one or more electrons in a molecule to higher energy levels. As this jump occurs, the electron may lose a small percentage of the absorbed energy (e.g., via collisons with other molecules, etc). As its electrons return from higher energy levels to ground state, the excited molecule can radiate energy. The energy generated in this way is, however, less than that originally involved in exciting the molecule. As a result, the wavelength of the light emitted (here, fluorescent light) is longer than that of the light used to excite the molecule. Emitted light energy can be detected using standard equipment, such as a detector positioned at a right angle to the incident light beam. S. Bakerman (ed.), Chem Cues: Fluorescence Polarization Immunoassay, Laboratory Management, 16-18 (July 1983); D. Freifelder, Fluorescence Spectroscopy, In: Physical Biochemistry: Applications to Biochemistry and Molecular Biology (2d ed.), 537-572 (1982).
An understanding of FPIA also requires an understanding of polarized light. Ordinary light can be thought of as a number of electromagnetic waves, each in a single plane; each wave passes through the central axis or path of the light beam. Polarized light, however, is light in which only one wave plane occurs (the others having been eliminated or screened out). When a fluorescent molecule is oriented such that its dipoles lie in the same plane as the light waves, it absorbs the polarized light. As it returns to its ground state, the molecule emits light in the same plane.
Two additional factors of importance in FPIA are time-related. First, the fluorescence lifetime of the molecule being used must be considered. The lifetime is the interval between excitation of the molecule by a polarized light burst and emission by the molecule of a similar burst. Second, the rotational relaxation time of the molecule--the time necessary for an excited molecule to move out of alignment so that emitted polarized light is emitted in a direction different from its excitation. Small molecules (e.g., haptens) rotate rapidly in solution; their rotational relaxation times are shorter than molecular fluorescence lifetime. As a result, after having absorbed polarized light such small molecules become randomly oriented by the time a burst of polarized emitted light is obtained. Larger molecules (e.g., immunoglobulins) rotate relatively slowly and have rotational times longer than the typical fluorescence lifetime.
Fluorescence polarization measurements rely on the fact that polarized excitation radiation gives rise to polarized emission radiation if no molecular rotation of the fluorophore occurs. A fluorophore is a fluorescent molecule or a compound which has the property of absorbing light at one wavelength and emitting it at a longer wavelength. As described above, the fluorophore bound to a small molecular hapten experiences molecular rotation at a rate that is rapid compared to the lifetime of the excited state prior to emission. Thus, the light is depolarized when bound to a small molecule. When antibody binds the fluorophore-antigen, rotation decreases dramatically because of the large size (molecular weight - 150,000) of the antibody, causing the emitted light to remain polarized.
Immunoassays utilize this phenomenon as follows: with antibody and fluorophore-labeled hapten or antigen present, binding occurs between hapten or antibody and fluorophore-antigen and little fluorescence depolarization occurs. As antigen to be analyzed is added, it binds to antibody competitively, fluorophore antigen is not bound and depolarization is observed. The depolarization is a function of antigen concentration and constitutes a quantitative assay.
Fluorescence polarization has been widely used in the study of the interaction of small (fluorescent) molecules with proteins, (e.g., antibodies). Dandliker, W. B. et al., Immunochemistry, 1:165-191 (1964); Dandliker, W. B. and S. A. Levison, Immunochemistry, 5:171-183 (1967); Tengerdy, R. P., Journal of Laboratory and Clinical Medicine, 70:707-714 (1967). The first applications of the polarization principle in quantitative immunoassays were carried out in 1973 by Dandliker and Spencer et al. Dandliker, W. B. et al., Immunochemistry, 10:219-227 (1973); Spencer, R. D. et al., Clinical Chemistry, 19:838-844 (1973).
Although the principle of fluorescence polarization immunoassay (FPIA) had been known since the 1970's and feasible instrumentation for the assay with flow-cell and digital read-out has been available since 1973, FPIA has had relatively limited use clinically because it is limited in the size of analytes for whose detection it can be used. The FPIA method is simple, rapid and precise, but its sensitivity is limited. Because only a relatively small change in the polarization occurs, the method is not applicable to antigens whose molecular mass exceeds about 20,000 Daltons.